Project Summary Mark-release-recapture (MRR) studies are a standard method for estimating the entomological variables that govern the vectorial capacity of arthropod disease vectors. While these studies are a valuable means of estimating mosquito population density, length of gonotrophic cycle, population size, survivorship, and patterns of mosquito dispersal throughout the environment, technical and logistical challenges remain with existing methodologies. The most widely-used technique of topically-applying a fluorescent dust is limited by the necessity to release large numbers of mosquitoes, the potential impact of the marking technique on mosquito fitness, and the lack of persistence of the marks over time. More recently, trace elements and stable isotopes have provided a natural means of labeling mosquito larval habitat, but the use of multiple unique markers is limited and analytical methods involving mass spectrometry are typically cost-prohibitive. In this proposal, we will develop the novel application of marking mosquitoes with engineered protein microcrystals carrying unique DNA barcodes. Mosquitoes ingesting these crystals in the field as either larvae or adults are thus marked with a unique and information-rich DNA signature that can be amplified by PCR from surveillance pools. We hypothesize that both adult and larval mosquitoes can be persistently marked with unique DNA barcodes through ingestion of smart microcrystals. Unique barcodes are associated with georeferenced bait stations, thereby informing the location and time of mark acquisition, when mosquitoes are collected and sorted from surveillance traps. We will also integrate ex vivo pathogen biosensing capability into the crystals to further enhance surveillance applications. We hypothesize that DNA beacons installed at high concentration within host crystals can provide an amplified optical signal that reports on the pathogen infection status of field- collected mosquitoes. Projected outcomes include the optimization of barcode recovery from marked mosquitoes, the molecular engineering of DNA beacons targeting West Nile virus (WNV) into existing crystals, and validation of these beacons in vitro as well as in mosquito homogenates containing WNV. Further, we will optimize the delivery method of microcrystals to adult and larval mosquitoes, and determine tissue localization and persistence. During year 2, we will deploy smart microcrystals loaded with unique DNA barcodes to test their field capacity to report the seasonal movement patterns of Cx. tarsalis mosquitoes along habitat corridors. To date, we have demonstrated that DNA barcodes can be stored, recovered, and amplified from crystals by qPCR, that nullomer sequences used in barcode design do not amplify the DNA of Cx. tarsalis or Ae. aegypti mosquitoes (Fig 2b), and that adult mosquitoes can ingest smart microcrystals loaded with fluorescein, and subsequently the fluorescence can be detected using an in vivo imaging system (Fig 5). The proposed studies lay the groundwork for future large-scale field applications of this technology.